Part:BBa_K4361214

BlcR BTB primer F7.4 A62K

For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein (Part:BBa_K4361106) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as Rn with n being the number of the set) and a varying number of forward primers (denoted as Fn.m followed by the induced substitution). A single amino acid substitution is built-in in the forward primer to introduce a point mutation in the BlcR production plasmid (Part:BBa_K4361106).

The mutations are built-in with a site-directed mutagenesis technique. A DNA polymerase is used to lengthen the single-strand primer. After PCR, a Kinase, Ligase, and DpnI (KLD) mix reaction can be performed. The KLD mix contains three enzymes: kinase, for phosphorylation, ligase for ligation, and DpnI for removal of the plasmid template (Figure 1). DNA sequencing is used to identify mutants and determine whether they carry the desired mutation. To know more about the methods we used, see the protocols page.

Figure 1. Site-directed mutagenesis cycle. The first step is the design of the back-to-back primers with the substitution in the forward primer. Second is the amplification of the plasmid with the back-to-back primers to build in the mutation. The third is the phosphorylation by kinase, the ligation by ligase, and the template removal by DpnI..

The primer sets are divided as follows:

• Set 1, residues 36 to 39
  • Part:BBa_K4361200: R1
  • Part:BBa_K4361201: F1.1 D37R
  • Part:BBa_K4361202: F1.2 D37V
  • Part:BBa_K4361203: F1.3 L38V
• Set 4, residues 40 to 41
  • Part:BBa_K4361204: R4
  • Part:BBa_K4361205: F4.1 A40V
• Set 5, residues 47 to 49
  • Part:BBa_K4361206: R5
  • Part:BBa_K4361207: F5.1 T47S
  • Part:BBa_K4361208: F5.2 T47V
  • Part:BBa_K4361209: F5.3 A48V
• Set 7, residues 61 to 63
  • Part:BBa_K4361210: R7
  • Part:BBa_K4361211: F7.1 S61V
  • Part:BBa_K4361212: F7.2 A62V
  • Part:BBa_K4361213: F7.3 A62I
  • Part:BBa_K4361214: F7.4 A62K
  • Part:BBa_K4361215: F7.5 A62T
  • Part:BBa_K4361216: F7.6 H63V
  • Part:BBa_K4361217: F7.7 H63Y
• Set 3, residues 65 to 68
  • Part:BBa_K4361218: R3
  • Part:BBa_K4361219: F3.1 L66V
  • Part:BBa_K4361220: F3.2 L66A
  • Part:BBa_K4361221: F3.3 L66I
  • Part:BBa_K4361222: F3.4 A67Q
  • Part:BBa_K4361223: F3.5 A67V
  • Part:BBa_K4361224: F3.6 A67H
  • Part:BBa_K4361225: F3.7 V68T
  • Part:BBa_K4361226: F3.8 V68K
  • Part:BBa_K4361227: F3.9 V68S

This primer contains a mutation that changes the codon for alanine (A, GCG) in position 62 to that of lysine (K, AAA).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]